Most strategies for gene transfer in plants involve the introduction of foreign DNA into protoplasts to enable its integration into the nuclear genome (1-3). However, many of the economically important gene products (e.g., the protein conferring atrazine resistance) either are chloroplast encoded or, if they are nucleus encoded, are functional within the chloroplasts (e.g., enol-pyruvylshikimate-phosphate synthase, which confers resistance to glyphosate) (4) or mitochondria (e.g., aryl acylamidase, which confers resistance to propanil) (5, 6). Furthermore, the 1000-fold higher copy number of chloroplast genes relative to nuclear genes (7-9) makes feasible the introduction of multiple copies of foreign genes into plant cells, should the foreign genes become stably integrated into the chloroplast genome.
To obtain gene transfer into chloroplasts, the isolation of intact organelles capable of efficient uptake, transcription, and translation of foreign DNA is essential. As a first step towards achieving this goal, Daniell and Rebeiz isolated plastids from dark-growth cucumber cotyledons (etioplasts) capable of synthesis of protocholorophyllide (10) and chlorophyll (11-13) at extremely high rates. Also, etioplasts that had been loaded with prothylakoid proteins by treatment of etiolated cucumber cotyledons with hormones (14) converted prothylakoids into macrograna when illuminated in a cofactor-enriched medium (15). Daniell and colleagues also demonstrated the development of electron transport coupled to photophosphorylation in concordance with the synthesis of required polypeptides in isolated etioplasts (16, 17). Finally, they also observed linear biosynthesis of pigment and translation of endogenous messages for 8 hr. (18). These observations collectively establish that etioplasts of cucumber cotyledons are both metabolically very active and unusually stable in their capacity for protein synthesis, marking them as exceptional targets for gene incorporation and expression.